The present invention relates generally to diabetes mellitus, and in particular to compositions and methods for the diagnosis, prognosis and treatment of type 2 diabetes.
Type 2 diabetes mellitus, or xe2x80x9clate onsetxe2x80x9d diabetes, is a common, degenerative disease affecting 5 to 10 percent of the population in developed countries. The propensity for developing type 2 diabetes mellitus (xe2x80x9ctype 2 DMxe2x80x9d) is reportedly maternally inherited, suggesting a mitochondrial genetic involvement. (Alcolado, J. C. and Alcolado, R., Br. Med. J. 302:1178-1180 (1991); Reny, S. L., International J. Epidem. 23:886-890 (1994)). Diabetes is a heterogeneous disorder with a strong genetic component; monozygotic twins are highly concordant and there is a high incidence of the disease among first degree relatives of affected individuals.
Current pharmacological therapies for type 2 DM include injected insulin, and oral agents that are designed to lower blood glucose levels. Currently available oral agents include (i) the sulfonylureas, which act by enhancing the sensitivity of the pancreatic beta cell to glucose, thereby increasing insulin secretion in response to a given glucose load; (ii) the biguanides, which improve glucose disposal rates and inhibit hepatic glucose output; (iii) the thiazolidinediones, which improve peripheral insulin sensitivity through interaction with nuclear peroxisome proliferator-activated receptors (PPAR, see, e.g., Spiegelman, 1998 Diabetes 47:507-514; Schoonjans et al., 1997 Curr. Opin. Lipidol. 8:159-166; Staels et al., 1997 Biochimie 79:95-99), (iv) repaglinide, which enhances insulin secretion through interaction with ATP-dependent potassium channels; and (v) acarbose, which decreases intestinal absorption of carbohydrates.
At the cellular level, the degenerative phenotype that may be characteristic of late onset diabetes mellitus includes indicators of altered mitochondrial respiratory function, for example impaired insulin secretion, decreased ATP synthesis and increased levels of reactive oxygen species. Studies have shown that type 2 DM may be preceded by or associated with certain related disorders. For example, it is estimated that forty million individuals in the U.S. suffer from impaired glucose tolerance (IGT). Following a glucose load, ciruculating glucose concentrations in IGT patients rise to higher levels, and return to baseline levels more slowly, than in unaffected individuals. A small percentage of IGT individuals (5-10%) progress to non-insulin dependent diabetes (NIDDM) each year. This form of diabetes mellitus, type 2 DM, is associated with decreased release of insulin by pancreatic beta cells and a decreased end-organ response to insulin. Other symptoms of diabetes mellitus and conditions that precede or are associated with diabetes mellitus include obesity, vascular pathologies, peripheral and sensory neuropathies and blindness.
It is clear that none of the current pharmacological therapies corrects the underlying biochemical defect in type 2 DM. Neither do any of these currently available treatments improve all of the physiological abnormalities in type 2 DM such as impaired insulin secretion, insulin resistance and/or excessive hepatic glucose output. In addition, treatment failures are common with these agents, such that multi-drug therapy is frequently necessary.
Due to the strong genetic component of diabetes mellitus, the nuclear genome has been the main focus of the search for causative genetic mutations. However, despite intense effort, nuclear genes that segregate with diabetes mellitus are rare and include, for example, mutations in the insulin gene, the insulin receptor gene and the glucokinase gene. By comparison, although a number of altered mitochondrial genes that segregate with diabetes mellitus have been reported (see generally e.g., PCT/US95/04063), relationships amongst mitochondrial and extramitochondrial factors that contribute to cellular respiratory and/or metabolic activities as they pertain to diabetes remain poorly understood.
Clearly there is a need for improved diagnostic methods for early detection of a risk for developing type 2 DM, and for better therapeutics that are targeted to correct biochemical and/or metabolic defects responsible for this disease, regardless of whether such a defect underlying altered mitochondrial function may have mitochondrial or extramitochondrial origins. The present invention provides compositions and methods related to indicators of altered mitochondrial function that are useful for determining the risk and degree of progression of type 2 DM and for treating this disease, and offers other related advantages.
In one aspect, the present invention provides a method for identifying a risk for Type 2 diabetes in a human subject, comprising comparing the level of at least one indicator of altered mitochondrial function in a biological sample from the subject with a control sample; and therefrom identifying the risk for Type 2 diabetes.
It is another aspect of the invention to provide a method for determining a degree of disease progression in a human subject having Type 2 diabetes, comprising: comparing the level of at least one indicator of altered mitochondrial function in each of first and second biological samples, the first and second biological samples being obtained from the subject at a first time point and a second time point, respectively; and therefrom determining the degree of progression of Type 2 diabetes. In yet another aspect the invention provides a method of identifying an agent suitable for treating a human subject suspected of being at risk for having type 2 diabetes, comprising: comparing the level of at least one indicator of altered mitochondrial function in one or more biological samples obtained from the subject in the presence and absence of a candidate agent; and therefrom determining the suitability of the candidate agent for treating type 2 diabetes. In still another aspect of the invention, there is provided a method of determining the suitability of an agent for treating a subject suspected of being at risk for having type 2 diabetes, comprising: comparing the level of at least one indicator of altered mitochondrial function in a biological sample obtained from the subject before and after administering to the subject a candidate agent; and therefrom determining the suitability of the candidate agent for treating type 2 diabetes.
Turning to another aspect, the invention provides a method of determining the suitability of an agent for treating a human subject suspected of being at risk for having type 2 diabetes, comprising comparing the level of at least one indicator of altered mitochondrial function in at least one biological sample obtained from a plurality of subjects before and after administering to each of the subjects a candidate agent; and therefrom determining the suitability of the candidate agent for treating type 2 diabetes.
It is another aspect of the present invention to provide a method of stratifying human subjects according to type 2 diabetes subtypes, comprising: comparing the level of at least one indicator of altered mitochondrial function in at least one biological sample obtained from each of a plurality of subjects; and therefrom stratifying the subjects according to type 2 diabetes subtype. In yet another aspect the invention provides a method of stratifying human subjects according to type 2 diabetes subtypes, comprising: comparing the level of at least one indicator of altered mitochondrial function in a biological sample obtained from each of a plurality of subjects before and after administering to each of the subjects a candidate agent; and therefrom stratifying the subjects according to type 2 diabetes subtype.
According to certain embodiments within any of the above aspects of the invention, the indicator of altered mitochondrial function is a mitochondrial electron transport chain enzyme. In certain embodiments the step of comparing comprises measuring electron transport chain enzyme catalytic activity. In certain embodiments the step of measuring comprises determining enzyme activity per mitochondrion in the sample. In certain embodiments the step of measuring comprises determining enzyme activity per unit of protein in the sample. In certain embodiments the step of comparing comprises measuring electron transport chain enzyme quantity. In certain embodiments the step of measuring comprises determining enzyme quantity per mitochondrion in the sample. In certain embodiments the step of measuring comprises determining enzyme quantity per unit of protein in the sample. In certain embodiments the mitochondrial electron transport chain enzyme comprises at least one subunit of mitochondrial complex I. In certain embodiments the mitochondrial electron transport chain enzyme comprises at least one subunit of mitochondrial complex II. In certain embodiments the mitochondrial electron transport chain enzyme comprises at least one subunit of mitochondrial complex III. In certain embodiments the mitochondrial electron transport chain enzyme comprises at least one subunit of mitochondrial complex IV. In certain embodiments the at least one subunit of mitochondrial complex IV is COX1, COX2 or COX4. In certain embodiments the mitochondrial electron transport chain enzyme comprises at least one subunit of mitochondrial complex V. In certain embodiments the at least one subunit of a mitochondrial complex V is ATP synthase subunit 8 or ATP synthase subunit 6.
According to certain other embodiments of the above aspects of the invention, the indicator of altered mitochondrial function is a mitochondrial matrix component. In certain embodiments the indicator of altered mitochondrial function is a mitochondrial membrane component. In certain embodiments the mitochondrial membrane component is a mitochondrial inner membrane component. In certain embodiments the mitochondrial membrane component is adenine nucleotide translocator (ANT), voltage dependent anion channel (VDAC), malate-aspartate shuttle, calcium uniporter, UCP-1, UCP-2, UCP-3, a hexokinase, a peripheral benzodiazepine receptor, a mitochondrial intermembrane creatine kinase, cyclophilin D, a Bcl-2 gene family encoded polypeptide, tricarboxylate carrier or dicarboxylate carrier.
In certain embodiments the indicator of altered mitochondrial function is a Krebs cycle enzyme. In certain embodiments the step of comparing comprises measuring Krebs cycle enzyme catalytic activity. In certain embodiments the step of measuring comprises determining enzyme activity per mitochondrion in the sample. In certain embodiments the step of measuring comprises determining enzyme activity per unit of protein in the sample. In certain embodiments the step of comparing comprises measuring Krebs cycle enzyme quantity. In certain embodiments the step of measuring comprises determining enzyme quantity per mitochondrion in the sample. In certain embodiments the step of measuring comprises determining enzyme quantity per unit of protein in the sample. In certain embodiments the Krebs cycle enzyme is citrate synthase. In certain embodiments the Krebs cycle enzyme is aconitase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinyl-coenzyme A synthetase, succinate dehydrogenase, fumarase or malate dehydrogenase.
In certain other embodiments of the above aspects of the invention, the indicator of altered mitochondrial function is mitochondrial mass per cell in the sample. In certain embodiments mitochondrial mass is determined using a mitochondria selective agent. In certain embodiments mitochondrial mass is determined using nonylacridine orange. In certain embodiments mitochondrial mass is determined by morphometric analysis. In certain embodiments the indicator of altered mitochondrial function is the number of mitochondria per cell in the sample. In certain embodiments the step of comparing comprises measuring a mitochondrion selective reagent. In certain embodiments the mitochondrion selective reagent is fluorescent.
According to certain other embodiments of the above aspects of the invention, the indicator of altered mitochondrial function is a co-predictor of altered mitochondrial function comprising the amount of mitochondrial DNA per cell in the sample and the step of comparing further comprises comparing at least one additional indicator of altered mitochondrial function. In certain embodiments the step of comparing comprises measuring mitochondrial DNA by contacting a biological sample containing mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in the mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of the primer to the mitochondrial DNA; and detecting hybridization of the primer to the mitochondrial DNA, and therefrom quantifying the mitochondrial DNA. In certain embodiments the step of detecting comprises a technique that may be polymerase chain reaction, oligonucleotide primer extension assay, ligase chain reaction, or restriction fragment length polymorphism analysis. In certain embodiments the step of comparing comprises measuring mitochondrial DNA by contacting a sample containing amplified mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in the amplified mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of the primer to the mitochondrial DNA; and detecting hybridization of the primer to the mitochondrial DNA, and therefrom quantifying the mitochondrial DNA. In certain embodiments the step of detecting comprises a technique that may be polymerase chain reaction, oligonucleotide primer extension assay, ligase chain reaction, or restriction fragment length polymorphism analysis. In certain embodiments the mitochondrial DNA is amplified using a technique that may be polymerase chain reaction, transcriptional amplification systems or self-sustained sequence replication. In certain embodiments the step of comparing comprises measuring mitochondrial DNA by contacting a biological sample containing mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in the mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of the primer to the mitochondrial DNA; and detecting hybridization and extension of the primer to the mitochondrial DNA to produce a product, and therefrom quantifying the mitochondrial DNA. In certain embodiments the step of comparing comprises measuring mitochondrial DNA by contacting a sample containing amplified mitochondrial DNA with an oligonucleotide primer having a nucleotide sequence that is complementary to a sequence present in the amplified mitochondrial DNA, under conditions and for a time sufficient to allow hybridization of the primer to the mitochondrial DNA; and detecting hybridization and extension of the primer to the mitochondrial DNA to produce a product, and therefrom quantifying the mitochondrial DNA. In certain embodiments the mitochondrial DNA is amplified using a technique that may be polymerase chain reaction, transcriptional amplification systems or self-sustained sequence replication. In certain embodiments the amount of mitochondrial DNA in the sample is determined using an oligonucleotide primer extension assay.
In certain embodiments of any of the above aspects of the invention, the indicator of altered mitochondrial function is the amount of ATP per cell in the sample. In certain embodiments the step of comparing comprises measuring the amount of ATP per mitochondrion in the sample. In certain embodiments the step of comparing comprises measuring the amount of ATP per unit protein in the sample. In certain embodiments the step of comparing comprises measuring the amount of ATP per unit mitochondrial mass in the sample. In certain embodiments the step of comparing comprises measuring the amount of ATP per unit mitochondrial protein in the sample. In certain embodiments the indicator of altered mitochondrial function is the rate of ATP synthesis in the sample. In certain embodiments the indicator of altered mitochondrial function is an ATP biosynthesis factor. In certain embodiments the step of comparing comprises measuring ATP biosynthesis factor catalytic activity. In certain embodiments the step of measuring comprises determining ATP biosynthesis factor activity per mitochondrion in the sample. In certain embodiments the step of measuring comprises determining ATP biosynthesis factor activity per unit mitochondrial mass in the sample. In certain embodiments the step of measuring comprises determining ATP biosynthesis factor activity per unit of protein in the sample. In certain embodiments the step of comparing comprises measuring ATP biosynthesis factor quantity. In certain embodiments the step of measuring comprises determining ATP biosynthesis factor quantity per mitochondrion in the sample. In certain embodiments the step of measuring comprises determining ATP biosynthesis factor quantity per unit of protein in the sample.
In certain embodiments of any of the above aspects of the present invention, the indicator of altered mitochondrial function is free radical production. In certain embodiments the indicator of altered mitochondrial function is reactive oxygen species, protein nitrosylation, protein carbonyl modification, DNA oxidation, mtDNA oxidation, protein oxidation, protein carbonyl modification, malondialdehyde adducts of proteins, a glycoxidation product, a lipoxidation product, 8xe2x80x2-OH-guanosine adducts or TBARS. In certain embodiments the indicator of altered mitochondrial function is reactive oxygen species. In certain embodiments the indicator of altered mitochondrial function is protein nitrosylation. In certain embodiments the indicator of altered mitochondrial function is DNA oxidation. In certain embodiments the indicator of altered mitochondrial function is mitochondrial DNA oxidation. In certain embodiments the indicator of altered mitochondrial function is protein carbonyl modification.
In yet other certain embodiments of any of the above aspects of the instant invention, the indicator of altered mitochondrial function is a cellular response to elevated intracellular calcium. In certain other embodiments the indicator of altered mitochondrial function is a cellular response to at least one apoptogen. In certain other embodiments the at least one indicator of altered mitochondrial function is a co-indicator of altered mitochondrial function and the step of comparing further comprises comparing at least one additional non-enzyme indicator of altered mitochondrial function. In certain embodiments the at least one additional non-enzyme indicator of altered mitochondrial function is a level of mitochondrial protein in the sample. In certain embodiments the co-indicator of altered mitochondrial function is citrate synthase, hexokinase II, cytochrome c oxidase, phosphofructokinase, glyceraldehyde phosphate dehydrogenase, glycogen phosphorylase, creatine kinase, NADH dehydrogenase, glycerol 3-phosphate dehydrogenase, triose phosphate dehydrogenase or malate dehydrogenase.
Turning to another aspect, the invention provides a method of treating a human patient having type 2 diabetes mellitus, comprising administering to the patient an agent that substantially restores to a normal level at least one indicator of altered mitochondrial function. In certain embodiments the indicator of altered mitochondrial function is a mitochondrial electron transport chain enzyme, a Krebs cycle enzyme, a mitochondrial matrix component, a mitochondrial membrane component or an ATP biosynthesis factor. In certain embodiments the indicator of altered mitochondrial function is mitochondrial number per cell or mitochondrial mass per cell. In certain embodiments the indicator of altered mitochondrial function is an ATP biosynthesis factor. In certain embodiments the indicator of altered mitochondrial function is the amount of ATP per mitochondrion, the amount of ATP per unit mitochondrial mass, the amount of ATP per unit protein or the amount of ATP per unit mitochondrial protein. In certain embodiments the indicator of altered mitochondrial function comprises free radical production. In certain embodiments the indicator of altered mitochondrial function comprises a cellular response to elevated intracellular calcium. In certain embodiments the at least one indicator of altered mitochondrial function is a co-indicator of altered mitochondrial function. In certain embodiments the co-indicator of altered mitochondrial function is citrate synthase, hexokinase II, cytochrome c oxidase, phosphofructokinase, glyceraldehyde phosphate dehydrogenase, glycogen phosphorylase, creatine kinase, NADH dehydrogenase, glycerol 3-phosphate dehydrogenase, triose phosphate dehydrogenase or malate dehydrogenase. In certain embodiments the at least one indicator of altered mitochondrial function is a co-predictor of altered mitochondrial function. In certain embodiments the co-predictor of altered mitochondrial function is an amount of mitochondrial DNA per cell in the patient.
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth herein which describe in more detail certain aspects of this invention, and are therefore incorporated by reference in their entireties.